Chemical cross-linking in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a powerful tool to study non-covalent protein complexes. Nevertheless, there are still many questions to answer: Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? In order to answer these questions, we performed systematic cross-linking studies with two complexes using the N8
hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): i) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a KD range of 30 nM to > 25 μM and ii) α-thrombin and basic pancreatic trypsin inhibitor (BPTI), which shows a buffer dependent KD value between 100 and 320 μM. Samples were analyzed by MALDI-MS. For NCoA-1•STAT6Y, a good correlation of the amount of cross-linked species with the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. The specificity of complex formation for the mid-affinity range up to about KD ≈ 25 μM could be proven by comparing against a non-binding peptide and by studying the concentration dependence. In order to study in which affinity range specific cross-linking can be applied, the weak α-thrombin•BPTI complex was investigated. Although variations of the sodium concentration can change the dissociation constant up to 3-fold for this interaction, no significant effect on the amount of detected complex was observed at different peptide concentrations. Our interpretation of this result is that the detected complex is not specific, but a nonspecifically cross-linked species. Consequently, chemical cross-linking is not applicable to low-affinity complexes with KD >> 25 μM with the experimental approach used in this study.